Fibroblast activation protein-α, a stromal cell surface protease, shapes key features of cancer associated fibroblasts through proteome and degradome alterations.

Cancer associated fibroblasts (CAFs) constitute an abundant stromal component of most solid tumors. Fibroblast activation protein (FAP) α is a cell surface protease that is expressed by CAFs. We corroborate this expression profile by immunohistochemical analysis of colorectal cancer specimens. To better understand the tumor-contextual role of FAPα, we investigate how FAPα shapes functional and proteomic features of CAFs using loss- and gain-of function cellular model systems. FAPα activity has a strong impact on the secreted CAF proteome ("secretome"), including reduced levels of anti-angiogenic factors, elevated levels of transforming growth factor (TGF) ß, and an impact on matrix processing enzymes. Functionally, FAPα mildly induces sprout formation by human umbilical vein endothelial cells. Moreover, loss of FAPα leads to a more epithelial cellular phenotype and this effect was rescued by exogenous application of TGFß. In collagen contraction assays, FAPα induced a more contractile cellular phenotype. To characterize the proteolytic profile of FAPα, we investigated its specificity with proteome-derived peptide libraries and corroborated its preference for cleavage carboxy-terminal to proline residues. By "terminal amine labeling of substrates" (TAILS) we explored FAPα-dependent cleavage events. Although FAPα acts predominantly as an amino-dipeptidase, putative FAPα cleavage sites in collagens are present throughout the entire protein length. In contrast, putative FAPα cleavage sites in non-collagenous proteins cluster at the amino-terminus. The degradomic study highlights cell-contextual proteolysis by FAPα with distinct positional profiles. Generally, our findings link FAPα to key aspects of CAF biology and attribute an important role in tumor-stroma interaction to FAPα.

Human Eb peptide: not just a by-product of pre-pro-IGF1b processing?

Several physiological activities have been assigned to E-peptides derived from pre-pro-insulin-like growth factor (IGF1) processing; however, the whole range of the E-peptides' functions is still unknown. The objective of this study was to investigate human Eb peptide (hEb) in terms of its bioactivity, cellular localization, and intracellular trafficking using human cancer cells. Human Eb fused with red fluorescence protein (RFP) or green fluorescence protein (GFP) localizes strongly to nucleoli and to a lesser extent to nuclei of HeLa and U2-OS cells. Mutagenesis of hEb nucleolus localization sequence (NoLS) leads to its partial delocalization from nuclei and nucleoli to cytoplasm of transfected cells. Thus, NoLS is not sufficient for the hEb to be localized in nucleoli of the cells and a different mechanism may be involved in hEb targeting. A BrdU ELISA showed that the proliferation index of cells expressing hEb hybrid proteins increased up to 28%. For comparison, the same assay was performed using HeLa cells treated extracellularly with synthetic hEb. A significant increase in the proliferation index was observed (41-58% for concentrations ranging from 10-100 nM, respectively). Additionally, a cell migration assay was performed using stable U2-OS cell lines expressing hEb fused with RFP or RFP alone as a negative control. The migration index of hEb expressing cells was 38.3% greater. The increase in cell proliferation index and in motile properties of hEb expressing cells demonstrate that hEb is more than a pre-pro-IGF1b processing product, and has intrinsic activity of biological significance.